We plan to continue our studies on the radial distribution of the cross bridges of thick filaments in the resisting and rigor state of glycerinated muscle using bifunctional reagents. Fibers of differing sarcomere lengths will be crosslinked in the presence of metabolites (Ca2ion, MgATP, MgADP) important in muscle contraction and the time course of crosslinking the myosin heads to the thick filament surface determined to establish under which conditions, if any, the cross-bridges leave the thick filament surface. We will also explore the effects of Ca2 ion on the hydrodynamic properties of native and crosslinked thick filaments under ionic conditions identical to those employed for the measurement of tension vs pCa curves of native (skinned) fibers. These experiments are relevant to cross-bridge movement and orientation when muscle is stimulated to contract. Further studies on the mechanism of formation of the thick filament are planned. The crosslinked myosin dimer will be isolated by zonal centrifugation, its geometry established by electron microscopy and its role in the assembly of the thick filament investigated. We also plan to continue our investigations on the role of two essential thiol groups in the activation mechanism of myosin. Our recent studies demonstrate that reconsituted actomyosin threads formed from highly polymerized vertebrate F-actin and containing only trace amounts of myosin show rapid shrinkage on addition of MgATP. We hope to determine the minimum size of the myosin species (monomer, dimer, bipolar filament) which is required for contraction in these systems by crosslinking the species within the threads at various levels of contraction. The role of the tail segment of myosin in the contractile mechanism of synthetic threads will be investigated through the use of hybrid threads reconstituted from mixtures of native myosin and heavy meromyosin. These studies are relevant to the mechanism of motile processes in non-muscle cells.